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- PDB-7qhb: Active state of GluA1/2 in complex with TARP gamma 8, L-glutamate... -

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Basic information

Entry
Database: PDB / ID: 7qhb
TitleActive state of GluA1/2 in complex with TARP gamma 8, L-glutamate and CTZ
Components
  • (Isoform Flip of Glutamate receptor ...) x 2
  • Voltage-dependent calcium channel gamma-8 subunit
KeywordsMEMBRANE PROTEIN / glutamate / AMPA receptor / TARPs
Function / homology
Function and homology information


Phase 0 - rapid depolarisation / Phase 2 - plateau phase / Cargo concentration in the ER / axonal spine / positive regulation of locomotion involved in locomotory behavior / COPII-mediated vesicle transport / positive regulation of membrane potential / cellular response to ammonium ion / response to sucrose / L-type voltage-gated calcium channel complex ...Phase 0 - rapid depolarisation / Phase 2 - plateau phase / Cargo concentration in the ER / axonal spine / positive regulation of locomotion involved in locomotory behavior / COPII-mediated vesicle transport / positive regulation of membrane potential / cellular response to ammonium ion / response to sucrose / L-type voltage-gated calcium channel complex / myosin V binding / LGI-ADAM interactions / neuron spine / Trafficking of AMPA receptors / postsynaptic neurotransmitter receptor diffusion trapping / proximal dendrite / regulation of AMPA receptor activity / protein phosphatase 2B binding / response to arsenic-containing substance / regulation of monoatomic ion transmembrane transport / channel regulator activity / cellular response to L-glutamate / ligand-gated calcium channel activity / cellular response to dsRNA / dendritic spine membrane / beta-2 adrenergic receptor binding / Synaptic adhesion-like molecules / long-term synaptic depression / cellular response to peptide hormone stimulus / spine synapse / dendritic spine neck / dendritic spine cytoplasm / dendritic spine head / cellular response to amine stimulus / peptide hormone receptor binding / response to psychosocial stress / Activation of AMPA receptors / ligand-gated monoatomic cation channel activity / response to morphine / perisynaptic space / spinal cord development / neuronal cell body membrane / protein kinase A binding / Trafficking of GluR2-containing AMPA receptors / response to lithium ion / AMPA glutamate receptor activity / AMPA glutamate receptor clustering / transmission of nerve impulse / kainate selective glutamate receptor activity / immunoglobulin binding / adenylate cyclase binding / behavioral response to pain / AMPA glutamate receptor complex / regulation of receptor recycling / response to electrical stimulus / extracellularly glutamate-gated ion channel activity / cellular response to glycine / ionotropic glutamate receptor complex / asymmetric synapse / Unblocking of NMDA receptors, glutamate binding and activation / G-protein alpha-subunit binding / glutamate receptor binding / conditioned place preference / positive regulation of synaptic transmission / regulation of postsynaptic membrane neurotransmitter receptor levels / long-term memory / voltage-gated calcium channel activity / postsynaptic density, intracellular component / response to fungicide / regulation of synaptic transmission, glutamatergic / neuronal action potential / extracellular ligand-gated monoatomic ion channel activity / cytoskeletal protein binding / glutamate-gated receptor activity / cellular response to brain-derived neurotrophic factor stimulus / regulation of long-term synaptic depression / positive regulation of synaptic transmission, glutamatergic / somatodendritic compartment / glutamate-gated calcium ion channel activity / presynaptic active zone membrane / synapse assembly / excitatory synapse / ionotropic glutamate receptor signaling pathway / ionotropic glutamate receptor binding / dendrite membrane / dendrite cytoplasm / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / positive regulation of excitatory postsynaptic potential / dendritic shaft / SNARE binding / synaptic membrane / PDZ domain binding / calcium channel regulator activity / response to cocaine / neuromuscular junction / establishment of protein localization / protein tetramerization / cellular response to amino acid stimulus / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic transmission, glutamatergic
Similarity search - Function
Butyryl-CoA Dehydrogenase, subunit A; domain 3 - #150 / Voltage-dependent calcium channel, gamma-8 subunit / : / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / Butyryl-CoA Dehydrogenase, subunit A; domain 3 / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel ...Butyryl-CoA Dehydrogenase, subunit A; domain 3 - #150 / Voltage-dependent calcium channel, gamma-8 subunit / : / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / Butyryl-CoA Dehydrogenase, subunit A; domain 3 / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / : / Ionotropic glutamate receptor / Eukaryotic homologues of bacterial periplasmic substrate binding proteins. / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
(2S)-2,3-dihydroxypropyl (7Z)-hexadec-7-enoate / CYCLOTHIAZIDE / GLUTAMIC ACID / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / PALMITOLEIC ACID / Glutamate receptor 1 / Glutamate receptor 2 / Voltage-dependent calcium channel gamma-8 subunit
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHerguedas, B. / Kohegyi, B. / Zhang, D. / Greger, I.H.
Funding support Spain, 3items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105174197 Spain
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N002113/1 Spain
Spanish Ministry of Science, Innovation, and UniversitiesPID2019-106284GA-I00 Spain
CitationJournal: Nat Commun / Year: 2022
Title: Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor.
Authors: Beatriz Herguedas / Bianka K Kohegyi / Jan-Niklas Dohrke / Jake F Watson / Danyang Zhang / Hinze Ho / Saher A Shaikh / Remigijus Lape / James M Krieger / Ingo H Greger /
Abstract: AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel ...AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop ('β1'), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.
History
DepositionDec 11, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 23, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-13969
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Isoform Flip of Glutamate receptor 1
B: Isoform Flip of Glutamate receptor 2
C: Isoform Flip of Glutamate receptor 1
D: Isoform Flip of Glutamate receptor 2
I: Voltage-dependent calcium channel gamma-8 subunit
J: Voltage-dependent calcium channel gamma-8 subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)492,70734
Polymers484,9706
Non-polymers7,73728
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Isoform Flip of Glutamate receptor ... , 2 types, 4 molecules ACBD

#1: Protein Isoform Flip of Glutamate receptor 1 / GluR-1 / AMPA-selective glutamate receptor 1 / GluR-A / GluR-K1 / Glutamate receptor ionotropic / ...GluR-1 / AMPA-selective glutamate receptor 1 / GluR-A / GluR-K1 / Glutamate receptor ionotropic / AMPA 1 / GluA1


Mass: 102661.930 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria1, Glur1 / Plasmid: PRK5 / Production host: Homo sapiens (human) / References: UniProt: P19490
#2: Protein Isoform Flip of Glutamate receptor 2 / GluR-2 / AMPA-selective glutamate receptor 2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / ...GluR-2 / AMPA-selective glutamate receptor 2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / AMPA 2 / GluA2


Mass: 96247.055 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Production host: Homo sapiens (human) / References: UniProt: P19491

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Protein , 1 types, 2 molecules IJ

#3: Protein Voltage-dependent calcium channel gamma-8 subunit / Neuronal voltage-gated calcium channel gamma-8 subunit / Transmembrane AMPAR regulatory protein ...Neuronal voltage-gated calcium channel gamma-8 subunit / Transmembrane AMPAR regulatory protein gamma-8 / TARP gamma-8


Mass: 43576.004 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Cacng8 / Production host: Homo sapiens (human) / References: UniProt: Q8VHW5

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Non-polymers , 5 types, 28 molecules

#4: Chemical
ChemComp-PAM / PALMITOLEIC ACID


Mass: 254.408 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C16H30O2
#5: Chemical
ChemComp-GLU / GLUTAMIC ACID


Type: L-peptide linking / Mass: 147.129 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C5H9NO4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-CYZ / CYCLOTHIAZIDE / 3-BICYCLO[2.2.1]HEPT-5-EN-2-YL-6-CHLORO-3,4- DIHYDRO-2H-1,2,4-BENZOTHIADIAZINE-7-SULFONAMIDE 1,1 DIOXIDE


Mass: 389.878 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H16ClN3O4S2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H40O4
#8: Chemical
ChemComp-79N / (2S)-2,3-dihydroxypropyl (7Z)-hexadec-7-enoate


Mass: 328.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C19H36O4

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex between GluA1/2 AMPA receptor and auxiliary subunit TARP gamma8
Type: COMPLEX
Details: GluA2 and TARP8 are expressed as a tandem construct
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.490 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Plasmid: PRK5
Buffer solutionpH: 8
Details: 25 mM TRIS 150 mM NaCl 0.02 % GDN 87 uM CTZ 100 mM L-Glu
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was incubated with 87 uM CTZ for 30 minutes and 100 mM L-Glutamate was added before grid preparation
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3-4 second blots

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 340 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4 sec. / Electron dose: 51 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9664

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Processing

SoftwareName: PHENIX / Version: 1.18.1_3865: / Classification: refinement
EM software
IDNameCategoryDetails (eV)
1RELIONparticle selection
2EPUimage acquisition
4GctfCTF correctionGctf was used to performed CTF correction
5RELIONCTF correctionCTF refinement was performed with relion
8UCSF Chimeramodel fitting
9Cootmodel fitting
11PHENIXmodel refinement
12REFMACmodel refinement
13RELIONinitial Euler assignment
14RELIONfinal Euler assignment
15RELIONclassification
16RELION3D reconstruction
CTF correctionDetails: CTF refinement was performed after 3D reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83344 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingDetails: Initial fitting was performed in Chimera with the Rigid Body fit option. The crystal structure of the L-Glu+CTZ GluA2 LBD was used for the LBD layer. After rigid body fitting Refmac and ...Details: Initial fitting was performed in Chimera with the Rigid Body fit option. The crystal structure of the L-Glu+CTZ GluA2 LBD was used for the LBD layer. After rigid body fitting Refmac and Phenix were used in refinement. Manual model building was performed in Coot
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16QKC

6qkc

16QKC1PDBexperimental model
23TKD

3tkd

13TKD2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01815366
ELECTRON MICROSCOPYf_angle_d0.99620786
ELECTRON MICROSCOPYf_dihedral_angle_d16.4772386
ELECTRON MICROSCOPYf_chiral_restr0.0672394
ELECTRON MICROSCOPYf_plane_restr0.0062548

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